Microneedle patch-based enzyme-linked immunosorbent assay to quantify protein biomarkers of tuberculosis.

There is a clinical need for differential diagnosis of the latent versus active stages of tuberculosis (TB) disease by a simple-to-administer test. Alpha-crystallin (Acr) and early secretory antigenic target-6 (ESAT-6) are protein biomarkers associated with the latent and active stages of TB, respectively, and could be used for differential diagnosis. We therefore developed a microneedle patch (MNP) designed for application to the skin to quantify Acr and ESAT-6 in dermal interstitial fluid by enzyme-linked immunosorbent assay (ELISA). We fabricated mechanically strong microneedles made of polystyrene and coated them with capture antibodies against Acr and ESAT-6. We then optimized assay sensitivity to achieve a limit of detection of 750 pg/ml and 3,020 pg/ml for Acr and ESAT-6, respectively. This study demonstrates the feasibility of an MNP-based ELISA for differential diagnosis of latent TB disease.

A retrospective cohort study assessing geometrical parameters related to visual deterioration in pituitary macroadenoma.

PURPOSE: We aim to identify specific parameters that will allow identification of those patients at greater risk of vision loss, specifically variables such as geometry, volume and size of the pituitary tumour. METHODOLOGY: Volume measurements were made with both manual (using the formula width × height × length × 0.5) and video size, sub-voxel interpolation using Brainlab software. The shape of the tumour was divided into four categories, ovoid, dumbbell, pyramidal and other. Chiasmal compression was divided into central and lateral compression and the chiasm location was classified based on pre/post and normal fixed chiasm in relation to the tuberculum sella. We used a visual impairment score ranging from (0-100), which was then further subdivided in to four grades: Grade I - 0-25 (mild); Grade II - 26-50 (moderate); Grade III - 51-75 (severe); Grade IV - 76-100 (subtotal or complete). RESULTS: Univariate analysis of variables related to vision loss found tumour volume, suprasellar growth and lateral chiasmal compression related to pre-operative vision loss. On multivariate regression analysis tumour volume and lateral chiasmal compression remained significant (p < 0.008). In relation to post-operative vision, univariate analysis indicated that variables such as giant macroadenoma, pre-operative visual impairment score and suprasellar growth were significant. Multivariate analysis showed pre-operative visual impairment and suprasellar growth as the only statistically significant variables. CONCLUSION: Our ROC analysis suggests tumours with suprasellar extension 16 mm (sensitivity 85 %, specificity 67 %) in sagittal plane are more likely to result in moderate to severe grade vision loss (VIS grade ≥ 2).

South African Lagerstätte reveals middle Permian Gondwanan lakeshore ecosystem in exquisite detail.

Continental ecosystems of the middle Permian Period (273-259 million years ago) are poorly understood. In South Africa, the vertebrate fossil record is well documented for this time interval, but the plants and insects are virtually unknown, and are rare globally. This scarcity of data has hampered studies of the evolution and diversification of life, and has precluded detailed reconstructions and analyses of ecosystems of this critical period in Earth's history. Here we introduce a new locality in the southern Karoo Basin that is producing exceptionally well-preserved and abundant fossils of novel freshwater and terrestrial insects, arachnids, and plants. Within a robust regional geochronological, geological and biostratigraphic context, this Konservat- and Konzentrat-Lagerstätte offers a unique opportunity for the study and reconstruction of a southern Gondwanan deltaic ecosystem that thrived 266-268 million years ago, and will serve as a high-resolution ecological baseline towards a better understanding of Permian extinction events.

Differential T-cell responses in dogs with meningoencephalomyelitis of unknown origin compared to healthy controls.

Meningoencephalomyelitis of unknown origin (MUO) is a common disorder in dogs that results in mononuclear inflammation in the brain and/or spinal cord. MUO is presumed to be autoimmune but specific immunological aberrations have not been identified. This exploratory study aimed to evaluate T cell production of two cytokines commonly implicated in autoimmune disease, interferon-gamma (IFNg) and interleukin-17 (IL17). Peripheral blood mononuclear cells were obtained from 12 dogs with MUO and 10 healthy controls, stimulated to activate intracellular signaling pathways, and stained with a cluster of differentiation 4 (CD4), cluster of differentiation eight (CD8), IFNg, and IL17 antibodies prior to analysis by flow cytometry. Mean differences in absolute cell numbers are represented as MUO cases minus healthy controls, and 95% Cis are reported. Overall IFNg-producing lymphocytes (mean difference = 241.8 cells/ul, 95% CI = 65.6 to 418.1) and CD4+ IFNg-producing T-cells (mean difference = 188.4, 95% CI = 77.3 to 299.5) were fewer in MUO cases. Additionally, CD4+ IL17-producing T-cells were greater in MUO cases (mean difference -34.9, 95% CI = -50.54 to -19.17) and CD8+ IL17-producing T-cells were fewer in MUO cases (mean difference = 73.5, 95% CI = 6.8 to 140.1). These results support that immunological changes can be identified in peripheral blood cells of dogs with MUO and suggest that T-helper type 17 (Th17) cells may play a role in pathogenesis.

Introduction of the day case total laparoscopic hysterectomy (TLH) protocol.

BACKGROUND: Traditionally total laparoscopic hysterectomy (TLH) patients are admitted for 1-2 days post-operatively. Day case TLH has been proven to be feasible and safe in other countries; however, this tertiary Queensland hospital is one of the first Australian institutions to introduce a day case TLH protocol. AIM: To pilot the implementation of our day case TLH protocol assessing the feasibility, safety and patient satisfaction of same-day discharge. MATERIALS AND METHODS: A retrospective audit of the implementation of our day case TLH protocol at a tertiary Queensland hospital was conducted. Primary outcome was length of post-operative hospital stay. Secondary outcomes included perioperative complications and post-operative re-presentation rates. Patient satisfaction was assessed through a patient questionnaire. RESULTS: Seventy-seven patients were included in the study. There were 94.81% patients who went home on the same day. Their average length of post-operative hospital stay was 7.72 (SD ± 3.36) hours. Of the patients who did achieve same-day discharge, the average length of stay was 7.05 (SD ±1.46) hours. There were no significant differences in perioperative complications or re-presentation rates compared to previously published literature. Patients reported they were extremely satisfied with day case TLH. CONCLUSION: The implementation of our day case TLH protocol is feasible, safe and well received by patients in our tertiary Australian hospital. These results can have multimodal effects in healthcare: decrease in hospital costs by reducing length of stay and overnight admissions, improved theatre efficiency and patient flow, while maintaining patient safety and satisfaction.

Neurosurgical trauma from E-Scooter usage: a review of early case series in London and a review of the literature.

BACKGROUND: Electric (E-)scooters have become increasingly popular as a means of environmentally friendly and convenient urban transport since 2017. Private use and now ride-sharing schemes are seen in major cities across the world. However, safety data are still lacking especially with concerning reports of significant head injuries. We describe early case series from three hospitals, including a major trauma centre, in East London and review the literature describing neurosurgical trauma from E-scooter usage. METHODS: Electronic health records were retrospectively searched for terms related to E-Scooters between 2018 and 2021. Eight case series were included and described. A literature search of PubMed, Ovid MEDLINE and Embase for terms 'E-scooter or electric scooter' was also conducted from inception to 6 March 2021. A total of 825 articles were initially reviewed. Following the removal of duplicates and those meeting the exclusion criteria, 29 articles were analysed in full and included in this review. RESULTS: All cases described a head injury of some nature with over half suffering more severe injuries including cranial fractures (most commonly basal skull) or intracerebral haemorrhages (ICH). Spinal fractures were also seen. All required imaging and admission to the hospital. Only a minority required inpatient neurosurgical intervention above conservative measures but almost all required outpatient follow-up. On review of the literature, head injuries were present in 38.8% of all presentations. The majority of which were minor head injuries or concussions; however, approximately 15% involved ICH or skull fractures, respectively. Spinal injuries were less common. Riding while intoxicated and without a helmet was frequently seen within the literature. CONCLUSION: This work offers a foundational text highlighting the frequency and severity of neurotrauma seen with E-scooter usage. Injury is seen in E-scooter riders, passengers and pedestrians alike with dangerous riding behaviours likely contributory. A neurosurgical-specific inquiry is recommended for future research.

Immunogenicity and protective efficacy of a pan-fungal vaccine in preclinical models of aspergillosis, candidiasis, and pneumocystosis.

Invasive fungal infections cause over 1.5 million deaths worldwide. Despite increases in fungal infections as well as the numbers of individuals at risk, there are no clinically approved fungal vaccines. We produced a "pan-fungal" peptide, NXT-2, based on a previously identified vaccine candidate and homologous sequences from Pneumocystis, Aspergillus,Candida, and Cryptococcus. We evaluated the immunogenicity and protective capacity of NXT-2 in murine and nonhuman primate models of invasive aspergillosis, systemic candidiasis, and pneumocystosis. NXT-2 was highly immunogenic and immunized animals had decreased mortality and morbidity compared to nonvaccinated animals following induction of immunosuppression and challenge with Aspergillus, Candida, or Pneumocystis. Data in multiple animal models support the concept that immunization with a pan-fungal vaccine prior to immunosuppression induces broad, cross-protective antifungal immunity in at-risk individuals.

Characterisation of the serum cytokine profile in feline atopic skin syndrome.

BACKGROUND: Feline atopic skin syndrome (FASS) is a pruritic and inflammatory skin disease commonly encountered in cats. Three previous reports evaluated cytokine immune activation in cats diagnosed with feline allergic dermatitis. However, no significant upregulations were observed in allergic cats compared to healthy controls. HYPOTHESIS/OBJECTIVE: To evaluate differences in the serum cytokine profile of cats diagnosed with FASS compared to healthy cats, and correlate serum markers with the extent of FASS skin disease using clinical scoring systems. ANIMALS: Thirteen client-owned FASS cats and 12 healthy control cats. METHODS AND MATERIALS: Thirteen cytokine and chemokines from the serum of FASS cats and healthy controls were analysed using a commercially available feline-specific multiplex assay. RESULTS: Patients with FASS had a significant increase in serum concentrations of interferon-gamma (IFN-γ), interleukin (IL)-2, IL-13 and IL-18. In addition, cytokine/chemokines involved in inflammation and chemotaxis [IL-8, C-C Motif Chemokine Ligand (CCL)5, CCL2 and CXCL12], as well as growth factors, stem cell factor and Fms-related tyrosine kinase 3 ligand (Flt3L), also were significantly elevated. A significant positive correlation (r = 0.64) between the serum levels of Flt3L and Scoring Feline Allergic Dermatitis (SCORFAD) score was observed. CONCLUSIONS: These results demonstrate the activation of a broad array of immune secretory cytokines in the serum of cats with FASS, which are largely associated with a mixed Th1 and Th2 inflammatory response along with specific growth factors. Further larger-sample studies are needed to assess the modulation of serum biomarkers in FASS by pharmacological/therapeutic interventions.

Contextualizing social class and leadership in fraternity and sorority communities.

This chapter examines leadership and social class in the context of fraternities and sororities. With no extensive research in this area, recommendations provided may help educators create a plan to address the intersection of social class, leadership education, and membership in a fraternity or sorority.

Solar-driven water-splitting provides a solution to the energy problem underpinning climate change.

The emergence of the oxygen-evolving photosystem two complex over 2.6 billion years ago represented the 'big bang of evolution' on planet Earth. It allowed phototrophic organisms to use sun light as an energy source to extract electrons and protons from water, and concomitantly release oxygen. Oxygenic photosynthesis not only created an aerobic atmosphere but also removed CO2 to produce the organic molecules that make up the current global biomass and fossil fuel. In addition, it paved the way for animal life. Today extensive burning of fossil fuels is reversing the results of photosynthesis through billions of years, rapidly releasing CO2 back into the atmosphere and consequently increasing the temperature of the planet. There is an urgent need to develop new sustainable energy sources, but the choice is not obvious. My approach to this problem has been to unravel the blueprint of photosystem II (PSII) and to develop an 'Artificial Leaf' technology. A significant step with respect to that mission was achieved at Imperial College when we could conclude from X-ray diffraction of PSII crystals, that the water-splitting catalytic centre consists of a unique Mn3Ca2+O4 cubane structure with a fourth dangler Mn oxo-bonded to the cubane. Here I use this and more recent structures to discuss the mechanism of water splitting and O-O bond formation. Furthermore, I will address how this information can be used to design novel water-splitting catalysts and highlight recent progress in this direction. My conviction is 'if plants can do it, we can do it - after all it is all about chemistry'.

A reassessment of the genus Oligoneuriopsis Crass, 1947 (Ephemeroptera, Oligoneuriidae, Oligoneuriellini).

The distinction between the two closely related genera Oligoneuriella Ulmer, 1924 and Oligoneuriopsis Crass, 1947 has been much debated. First described from South Africa, Oligoneuriopsis seemed to be a clearly defined genus. However, as the known distribution of the genus widened and knowledge on it expanded, species delimitation based on morphology became less clear due to overlap in several apparently defining morphological characters, especially in the nymphs. This work attempts to reassess Oligoneuriopsis morphology in the context of all currently known species. The type species, Oligoneuriopsis lawrencei Crass, 1947 is redescribed at the imaginal and nymphal stages and a neotype is designated. The putative nymph of Oligoneuriopsis dobbsi (Eaton, 1912) is described based on material collected around Mt Elgon (Kenya). The adults of Oligoneuriella orontensis Koch, 1980 are described for the first time and the species is transferred to the genus Oligoneuriopsis (Oligoneuriopsis orontensis comb. nov.). Egg structure is also described for the first time for the species Oligoneuriopsis skhounate and O. orontensis. Some biogeographical considerations are also given. It is likely that more species will still be discovered, especially in Africa.

Peripheral Tolerance Checkpoints Imposed by Ubiquitous Antigen Expression Limit Antigen-Specific B Cell Responses under Strongly Immunogenic Conditions.

A series of layered peripheral checkpoints maintain self-reactive B cells in an unresponsive state. Autoantibody production occurs when these checkpoints are breached; however, when and how this occurs is largely unknown. In particular, how self-reactive B cells are restrained during bystander inflammation in otherwise healthy individuals is poorly understood. A weakness has been the unavailability of methods capable of dissecting physiologically relevant B cell responses without the use of an engineered BCR. Resolving this will provide insights that decipher how this process goes awry during autoimmunity or could be exploited for therapy. In this study, we use a strong adjuvant to provide bystander innate and adaptive signals that promote B cell responsiveness in conjunction with newly developed B cell detection tools to study in detail the ways that peripheral tolerance mechanisms limit the expansion and function of self-reactive B cells activated under these conditions. We show that although self-reactive B cells are recruited into the germinal center, their development does not proceed, possibly because of rapid counterselection. Consequently, differentiation of plasma cells is blunted, and Ab responses are transient and devoid of affinity maturation. We propose this approach, and these tools can be more widely applied to track Ag-specific B cell responses to more disease-relevant Ags, without the need for BCR transgenic mice, in settings where tolerance pathways are compromised or have been genetically manipulated to drive stronger insights into the biology underlying B cell-mediated autoimmunity.

Transfer of antigen-encoding bone marrow under immune-preserving conditions deletes mature antigen-specific B cells in recipients and inhibits antigen-specific antibody production.

BACKGROUND AIMS: Pathological activation and collaboration of T and B cells underlies pathogenic autoantibody responses. Existing treatments for autoimmune disease cause non-specific immunosuppression, and induction of antigen-specific tolerance remains an elusive goal. Many immunotherapies aim to manipulate the T-cell component of T-B interplay, but few directly target B cells. One possible means to specifically target B cells is the transfer of gene-engineered BM that, once engrafted, gives rise to widespread specific and tolerogenic antigen expression within the hematopoietic system. METHODS: Gene-engineered bone marrow encoding ubiquitous ovalbumin expression was transferred after low-dose (300-cGy) immune-preserving irradiation. B-cell responsiveness was monitored by analyzing ovalbumin-specific antibody production after immunization with ovalbumin/complete Freund's adjuvant. Ovalbumin-specific B cells and their response to immunization were analyzed using multi-tetramer staining. When antigen-encoding bone marrow was transferred under immune-preserving conditions, cognate antigen-specific B cells were purged from the recipient's preexisting B-cell repertoire and the repertoire that arose after bone marrow transfer. RESULTS: OVA-specific B-cell deletion was apparent within the established host B-cell repertoire as well as that developing after gene-engineered bone marrow transfer. OVA-specific antibody production was substantially inhibited by transfer of OVA-encoding BM and activation of OVA-specific B cells, germinal center formation and subsequent OVA-specific plasmablast differentiation were all inhibited. Low levels of gene-engineered bone marrow chimerism were sufficient to limit antigen-specific antibody production. RESULTS: These data show that antigen-specific B cells within an established B-cell repertoire are susceptible to de novo tolerance induction, and this can be achieved by transfer of gene-engineered bone marrow. This adds further dimensions to the utility of antigen-encoding bone marrow transfer as an immunotherapeutic tool.

Habitat requirements affect genetic variation in three species of mayfly (Ephemeroptera, Baetidae) from South Africa.

This study investigates genetic diversity in three species of Ephemeroptera, one eurytopic and therefore widespread (Afroptilum sudafricanum) and two stenotopic and thus endemic (Demoreptus natalensis and Demoreptus capensis) species, all of which co-occur in the southern Great Escarpment, South Africa. Mitochondrial DNA was analysed to compare the genetic diversity between the habitat generalist and the two habitat specialists. Afroptilum sudafricanum showed no indication of population genetic structure due to geographic location, while both Demoreptus species revealed clear genetic differentiation between geographic localities and catchments, evident from phylogenetic analyses and high FST values from AMOVA. In addition, the phylogenetic analyses indicate some deeper haplotype divergences within A. sudafricanum and Demoreptus that merit taxonomic attention. These results give important insight into evolutionary processes occurring through habitat specialisation and population isolation. Further research and sampling across a wider geographic setting that includes both major mountain blocks of the Escarpment and lowland non-Escarpment sites will allow for refined understanding of biodiversity and associated habitat preferences, and illuminate comparative inferences into gene flow and cryptic speciation.

Pectin-Alginate Does Not Further Enhance Exogenous Carbohydrate Oxidation in Running.

PURPOSE: Maximizing carbohydrate availability is important for many endurance events. Combining pectin and sodium alginate with ingested maltodextrin-fructose (MAL + FRU + PEC + ALG) has been suggested to enhance carbohydrate delivery via hydrogel formation, but the influence on exogenous carbohydrate oxidation remains unknown. The primary aim of this study was to assess the effects of MAL + FRU + PEC + ALG on exogenous carbohydrate oxidation during exercise compared with a maltodextrin-fructose mixture (MAL + FRU). MAL + FRU has been well established to increase exogenous carbohydrate oxidation during cycling compared with glucose-based carbohydrates (MAL + GLU). However, much evidence focuses on cycling, and direct evidence in running is lacking. Therefore, a secondary aim was to compare exogenous carbohydrate oxidation rates with MAL + FRU versus MAL + GLU during running. METHODS: Nine trained runners completed two trials (MAL + FRU and MAL + FRU + PEC + ALG) in a double-blind, randomized crossover design. A subset (n = 7) also completed a MAL + GLU trial to address the secondary aim, and a water trial to establish background expired CO2 enrichment. Participants ran at 60% V˙O2peak for 120 min while ingesting either water only or carbohydrate solutions at a rate of 1.5 g carbohydrate per minute. RESULTS: At the end of 120 min of exercise, exogenous carbohydrate oxidation rates were 0.9 (SD 0.5) g·min with MAL + GLU ingestion. MAL + FRU ingestion increased exogenous carbohydrate oxidation rates to 1.1 (SD 0.3) g·min (P = 0.038), with no further increase with MAL + FRU + PEC + ALG ingestion (1.1 (SD 0.3) g·min; P = 1.0). No time-treatment interaction effects were observed for plasma glucose, lactate, insulin, or nonesterified fatty acids, or for ratings of perceived exertion or gastrointestinal symptoms (all, P > 0.05). CONCLUSION: To maximize exogenous carbohydrate oxidation during moderate-intensity running, athletes may benefit from consuming glucose(polymer)-fructose mixtures over glucose-based carbohydrates alone, but the addition of pectin and sodium alginate offers no further benefit.

Intrastriatal injection of preformed alpha-synuclein fibrils alters central and peripheral immune cell profiles in non-transgenic mice.

Parkinson's disease (PD) is characterized by the accumulation of alpha-synuclein (α-syn) inclusions, the major component of Lewy bodies. Extracellular α-syn aggregates act as a damage-associated molecular pattern (DAMP) and the presence of autoantibodies against α-syn species in the cerebrospinal fluid and the serum of PD patients implicate the involvement of innate and adaptive immune responses. In non-transgenic (Tg) mice, intrastriatal injection of preformed fibril (PFF) α-syn results in widespread pathologic α-syn inclusions in the CNS. While the PFF model has been broadly utilized to study the mechanistic relationship between α-syn transmission and other neuropathological phenotypes, the immune phenotypes in this model are not clearly demonstrated. This study aimed to characterize the immune phenotypes during pathologic α-syn propagation by utilizing PFF α-syn-injected non-tg mice. Here, we showed that pathologic α-syn inclusions are prevalent in various brain regions and the gut at 5 months post injection (p.i.), preceding the degeneration of dopaminergic neurons in substantia nigra (SN). We discovered a distinct inflammatory response involving both activation of microglia and astrocytes and infiltration of B, CD4+ T, CD8+ T, and natural killer cells in the brain at 5 months p.i. Moreover, PFF α-syn-injected mice display significant alterations in the frequency and number of leukocyte subsets in the spleen and lymph nodes with minimum alterations in the blood. Our data provide primary evidence that intracerebral-initiated synucleinopathies in non-tg mice alter immune cell profiles both in the CNS and peripheral lymphoid organs. Furthermore, our data provides support for utilizing this mouse model to assess the mechanistic connection between immune responses and synuclein pathology.

Precipitation of Soluble Uric Acid Is Necessary for In Vitro Activation of the NLRP3 Inflammasome in Primary Human Monocytes.

OBJECTIVE: To investigate the effects of soluble uric acid (UA) on expression and activation of the NOD-like receptor (NLR) pyrin domain containing protein 3 (NLRP3) inflammasome in human monocytes to elucidate the role of hyperuricemia in the pathogenesis of gout. METHODS: Primary human monocytes and the THP-1 human monocyte cell line were used to determine the effects of short- and longterm exposure to UA on activation of the NLRP3 inflammasome and subsequent interleukin 1ß (IL-1ß) secretion by ELISA and cell-based assays. Expression of key NLRP3 components in monocytes from patients with a history of gout were analyzed by quantitative PCR. RESULTS: Precipitation of UA was required for activation of the NLRP3 inflammasome and subsequent release of IL-1ß in human monocytes. Neither monosodium urate (MSU) crystals nor soluble UA had any effect on activation of the transcription factor, nuclear factor-κB. Prolonged exposure of monocytes to soluble UA did not alter these responses. However, both MSU crystals and soluble UA did result in a 2-fold increase in reactive oxygen species. Patients with gout (n = 15) had significantly elevated serum UA concentrations compared to healthy individuals (n = 16), yet secretion of IL-1ß and expression of NLRP3 inflammasome components in monocytes isolated from these patients were not different from those of healthy controls. CONCLUSION: Despite reports indicating that soluble UA can prime and activate the NLRP3 inflammasome in human peripheral blood mononuclear cells, precipitation of soluble UA into MSU crystals is essential for in vitro NLRP3 signaling in primary human monocytes.

Immunomodulatory in vitro effects of oclacitinib on canine T-cell proliferation and cytokine production.

BACKGROUND: Oclacitinib is a Janus kinase inhibitor used to control pruritus and skin lesions in canine allergic skin disease; its effect on canine T cells is not well-characterized. HYPOTHESIS/OBJECTIVES: To evaluate the impact of oclacitinib on cultured T cells using peripheral blood mononuclear cells from dogs. ANIMALS: Six bluetick coonhounds. METHODS AND MATERIALS: Lymphocyte-enriched cells were incubated with or without the T-cell mitogen concanavalin A (Con A), oclacitinib (0.5, 1 or 10 µM), ciclosporin (200 ng/mL), Con A + oclacitinib 1 µM and Con A + ciclosporin. We assessed both T-cell proliferation and the secretion of cytokines. RESULTS: Ciclosporin and oclacitinib both inhibited the spontaneous proliferation of T cells; this effect was significant only after incubation with oclacitinib at 10 µM. At this concentration, oclacitinib significantly reduced the spontaneous secretion of clonal activator cytokines [interleukin (IL)-2, IL-15], pro-inflammatory cytokines (interferon-gamma (IFN-γ), IL-18) and the regulatory cytokine IL-10; tumour necrosis factor alpha (TNF-α) and IL-6 cytokine production was mildly inhibited. After Con A stimulation, only T cells co-treated with ciclosporin achieved a significant proliferation inhibition and reduction of IL-2, IL-10, IL-15, IL-18, IFN-γ and TNF-α. Surprisingly, oclacitinib at 1 µM (337 ng/mL, corresponding to the oral dosage of 0.4-0.6 mg/kg) did not significantly affect Con A-stimulated T-cell proliferation nor cytokine production (IL-2, IL-10, IL-15, IL-18, IFN-γ and TNF-α). CONCLUSIONS: Although a limited number of dogs were investigated, these preliminary results suggest that oclacitinib appears to have immunosuppressive properties, but only at dosages above those used to treat allergic pruritus in dogs.

Sheep breed and shearing influences attraction and blood-feeding behaviour of Culicoides (Diptera: Ceratopogonidae) on a UK farm.

BACKGROUND: Culicoides biting midges (Diptera: Ceratopogonidae) are responsible for the biological transmission of arboviruses of international importance between ruminant livestock. These arboviruses include bluetongue virus (BTV) and Schmallenberg virus (SBV), which have emerged in unprecedented outbreaks in northern Europe. The impact of breed and shearing of sheep on Culicoides: host contact rates has not been investigated in detail and has the potential to influence arbovirus transmission and control measures employed to limit spread. METHODS: Attraction of Culicoides to Hartline and Hartline/Suffolk cross-breed sheep was compared using 224 drop trap collections over 22 nights and 181 catches from sheared or unsheared Hartline/Suffolk ewes were made over 17 nights to compare Culicoides activity and rates of blood engorgement. RESULTS: A total of 31,314 Culicoides was collected in the two trials and females of the subgenus Avaritia represented over 96.9% of individuals collected. Attraction to breed was dependent upon species of Culicoides and physiological status, with a significantly greater number of individuals collected on the cross-breed sheep. Shearing of sheep did not significantly increase or decrease the number of Culicoides attracted but increased the rate of successful engorgement. CONCLUSIONS: Both breed and shearing were shown to influence Culicoides biting rate on sheep. These data are useful in a direct context in understanding the likely impact of control measures against arboviruses including BTV and SBV and additionally in providing data from field-based studies to enable modelling exercises of arbovirus transmission and spread.

Blood-feeding, susceptibility to infection with Schmallenberg virus and phylogenetics of Culicoides (Diptera: Ceratopogonidae) from the United Kingdom.

BACKGROUND: Culicoides biting midges (Diptera: Ceratopogonidae) are responsible for the biological transmission of internationally important arboviruses of livestock. In 2011, a novel Orthobunyavirus was discovered in northern Europe causing congenital malformations and abortions in ruminants. From field studies, Culicoides were implicated in the transmission of this virus which was subsequently named Schmallenberg virus (SBV), but to date no assessment of susceptibility to infection of field populations under standardised laboratory conditions has been carried out. We assessed the influence of membrane type (chick skin, collagen, Parafilm M®) when offered in conjunction with an artificial blood-feeding system (Hemotek, UK) on field-collected Culicoides blood-feeding rates. Susceptibility to infection with SBV following blood-feeding on an SBV-blood suspension provided via either (i) the Hemotek system or via (ii) a saturated cotton wool pledglet was then compared. Schmallenberg virus susceptibility was defined by RT-qPCR of RNA extractions of head homogenates and related to Culicoides species and haplotype identifications based on the DNA barcode region of the mitochondrial cytochrome c oxidase 1 (cox1) gene. RESULTS: Culicoides blood-feeding rates were low across all membrane types tested (7.5% chick skin, 0.0% for collagen, 4.4% Parafilm M®, with 6029 female Culicoides being offered a blood meal in total). Susceptibility to infection with SBV through membrane blood-feeding (8 of 109 individuals tested) and pledglet blood-feeding (1 of 94 individuals tested) was demonstrated for the Obsoletus complex, with both C. obsoletus (Meigen) and C. scoticus Downes & Kettle susceptible to infection with SBV through oral feeding. Potential evidence of cryptic species within UK populations was found for the Obsoletus complex in phylogenetic analyses of cox1 DNA barcodes of 74 individuals assessed from a single field-site. CONCLUSIONS: Methods described in this study provide the means to blood-feed Palaearctic Culicoides for vector competence studies and colonisation attempts. Susceptibility to SBV infection was 7.3% for membrane-fed members of the subgenus Avaritia and 1.1% for pledglet-fed. Both C. obsoletus and C. scoticus were confirmed as being susceptible to infection with SBV, with potential evidence of cryptic species within UK Obsoletus complex specimens, however the implications of cryptic diversity in the Obsoletus complex on arbovirus transmission remains unknown.